1. Field of the Invention
This invention relates to the use of a glucosyltransferase gene in recombinant cells to produce a precursor of sophorolipid and of phytosteryl glucosides.
2. Description of the Prior Art
Sophorolipid is a glycolipid produced and secreted by several yeast species such as Candida apicola, C. bombicola, Rhodotorula bogoriensis, Starmerella bombicola, Wickerhamiella domericqiae, and C. batistae (see, Van Bogaert, et al. 2007. Appl. Microbiol. Biotechnol. 76:23-34; and Konishi, et al. 2008. J. Oleo Sci. 57:359-369). Sophorolipid contains a hydrophilic disaccharide sophorose (2-O-β-D-glucopyranosyl-β-D-glucopyranose), which may or may not be enzymatically acetylated in vivo at the 6′-(C-6′) or both C-6′ and C-6″. The hydrophobic portion of the molecule contains a hydroxy fatty acid, which is glycosidically linked to the C-2′ of the sophorose moiety. In this respect, sophorolipid differentiates itself from the synthetic alkylpolyglucosides which have an ester bond between the hydrophobic and hydrophilic moieties of the molecule. The carboxyl group of the hydroxy fatty acid moiety of sophorolipid may also be found esterified in vivo to the C-4″ of the sophorose, resulting in a lactone structure of the molecule. The proportion of the various forms of sophorolipid produced from a particular fermentation depends to certain degree on the yeast strain used, the fermentation substrates, and the growth conditions. It should be noted that R. bogoriensis produces sophorolipid containing mainly a 13-hydroxydocosanoic acid (13-OH—C22) moiety (see Esders and Light 1972. J. Lipid Res. 13:636-671; Nuñez, et al. 2004. Biotechnol. Lett. 26:1087-1093; and Ribeiro, et al. 2012. J. Chromatogr. B 899:72-80), while the other sophorolipid-producing yeast strains predominantly synthesize the glycolipid having a 17-hydroxyoctadecenoic acid (17-OH—C18:1) as its hydrophobic component. The structural versatility of sophorolipid affords the possibility of designing specific production systems to tailor make products suitably targeted for an intended application.
Sophorolipid is an important microbial product with immense potential for industrial applications (Solaiman, et al. 2004a. Informs 15:270-272). Its amphiphatic structure bestows an excellent surface-active property to the molecule. Surface-tension measurements of an aqueous solution of sophorolipid routinely yielded values of 30-40 mN/m (see, for example, Solaiman, et al. 2004b. Biotechnol. Lett. 26:1241-1245). The antimicrobial activity and various biomedical properties of sophorolipid have been extensively documented (Hardin, et al. 2007. J. Surg. Res. 142:314-319; Kim, et al. 2005. J. Microbiol. Biotechnol. 15:55-58; Kim, et al. 2002. J. Microbiol. Biotechnol. 12:235-241, Krivobok, et al. 1994. J. Agric. Food Chem. 42:1247-1250; Lang, et al. 1989. Fett Wiss. Technol. 91:363-366; and Shah, et al. 2005. Antimicrob. Agents Chemother. 49:4093-4100). The potential applications of sophorolipid in the food-industry arena, such as its use as a formulation ingredient to modulate the rheological and textural properties and as an inhibitor of biofilm-formation by food pathogens, have been proposed (Nitschke and Costa 2007. Trends Food Sci. Technol. 18:252-259). Kasture et al. (Langmuir 23:11409-11412) recently showcased the value of sophorolipid in the field of nanotechnology by demonstrating its use in the capping of cobalt nanoparticles, thereby improving its water stability and dispersive property. The individual structural components of sophorolipid, i.e., the sophorose (Suto and Tomita 2001. J. Biosci. Bioeng. 92:305-311) and the hydroxy fatty acid (Zerkowski and Solaiman 2006. J. Am. Oil Chemists' Soc. 83:621-628; Zerkowski and Solaiman 2007. J. Am. Oil Chemists' Soc. 84:463-471), are also valuable specialty chemicals when separated and isolated (Rau, et al. 2001. Ind. Crops Prod. 13:85-92).
With a myriad of potential applications envisioned and the high commercial value expected of the sophorolipid, research and development efforts have largely been aimed at increasing the production yield and reducing the cost of this glycolipid. In comparison, fundamental research to delineate the metabolic pathway and the genetics of sophorolipid biosynthesis is lacking. A notable exception is a series of pioneering studies on the enzymology of sophorolipid biosynthesis of R. bogoriensis (formerly Candida bogoriensis) conducted by Esders, Light and collaborators. They first identified two glucosyl- and one acetyl-transferase activities in R. bogoriensis (Esders and Light, 1972a. J. Biol. Chem. 247:1375-1386). The two glucosyltransferase activities (i.e., glucosyltransferases 1 and 2), which resisted various chromatographic attempts to separate them, catalyze the sequential addition of glucose units to 13-hydroxydocosanoic acid to form the final sophorolipid molecule by utilizing UDP-glucose as substrate (Breithaupt and Light, 1982. J. Biol. Chem. 257:9622-9628; Esders and Light, 1972a). An UDP-glucose:sterol glucosyltransferase, which catalyzes the transfer of the activated glucosyl group to ergosterol (an indigenous substrate) and cholesterol, was also identified in the course of their studies (Esders and Light, 1972b. J. Biol. Chem. 247:7494-7497). These investigators further identified an acetyltransferase activity that catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to a mono- or un-acetylated sophorolipid (Esders and Light 1972a). The enzyme exhibited low reactivity toward mono-glucopyranosyl-13-hydroxydocosanote substrate. This finding, coupled with the observation that the acetyltransferase activity only peaked at a later time of fermentation, suggested that the acetylation of sophorolipid occurred mainly after the complete synthesis of the di-glucopyranosyl-13-hydroxydocosanoate. Cutler and Light (Cutler and Light, 1979. J. Biol. Chem. 254:1944-1950; and Cutler and Light, 1982. Can. J. Microbiol. 28:223-230) reported that a low concentration of glucose substrate led to diminished glucosyltransferase activities, and a high glucose concentration was necessary for the synthesis of fatty acids having 20- and 22-carbon chain length found in sophorolipid of R. bogoriensis. Although the enzymatic aspect of sophorolipid biosynthesis in R. bogoriensis was well-illustrated by this earlier elegant research in Light's laboratory, the probable sophorolipid biosynthesis pathway of C. bombicola is only beginning to emerge through a series of recent molecular biological studies (Saerens et al. 2011a. FEMS Yeast Res. 11:123-132; Saerens et al. 2011b. Yeast 28:279-292; and Van Bogaert et al. 2009. FEMS Yeast Res. 9:610-617). Saerens et al. 2011a reported the identification of a glucosyltransferase gene, UGTA1, in C. bombicola. Through knocking-out the UGTA1 in C. bombicola, it was shown that sophorolipid synthesis was not detected (Saerens et al. 2011a). The enzymatic function of the UGTA1 gene, however, was not confirmed by a direct biochemical assay in the same way as the Gtf-1 gene-product in this invention. Thus the inability of UGTA1 knock-out mutant to synthesize sophorolipid could not be conclusively attributed to the absence of glucosyltransferase activity in the first step of the biosynthesis pathway of sophorolipids.
While elucidating the genetic system of sophorolipid biosynthesis in C. bombicola, the cloning of a lipid-glucosyltransferase gene (gtf-1) from C. bombicola occurred, and the DNA sequence was published (GenBank Accession FJ231291.1). The DNA sequence of gft-1 differs from the other known yeast and fungal glucosyltransferases, including those identified recently in C. bombicola (Saerens, et al. 2011a; and Saerens, et al. 2011b. Yeast 28:279-292). However various uses of gtf-1 have not been disclosed until now.